This project was initiated to investigate the life cycle and basic function of pulmonary epithelial cells in a well-defined in vitro situation. Such a culture offers a model system to investigate the mechanism of cell differentiation, as well as the repair of injury and the pathological response of cells to environmental toxic substances. We have achieved conditions for long-term culture of normal tracheal epithelial cells from rabbit and rat by supplementing culture medium with hormones instead of serum. These hormones and factors are insulin, epidermal growth factor, transferrin, and an extract fraction from bovine hypothalamus. We have shown further in rat tracheal cultures that cell growth and survival are dependent on fibroblast conditioned medium and collagen-coated substratum. Ultrastructural and histochemical analyses suggest the epithelial nature of cells grown in hormone supplemented defined condition. The addition of retinoids to rabbit tracheal cultures increases periodic acid-schiff stain positive cells and the synthesis and secretion of 3H-glucosamine labeled glycoprotein. Similar study is now carried out in isolated rabbit Clara cells. We have found that Clara cells are better maintained in culture in hormone supplemented medium than in serum condition.